EVIDENCE FOR A PHOSPHORYL-ENZYME INTERMEDIATE IN ALKALINE PHOSPHATASE CATALYZED REACTIONS

Kinetic evidence is presented which demonstrates the formation of a catalytic phosphoryl-enzyme intermediate during the hydrolysis of phosphate esters by alkaline phosphatase. Nine phosphate esters were hydrolyzed in the presence of 1 m Tris acting as a phosphate acceptor in competition with water and the ratios of the products were measured. Precisely 1.39 equiv of O-phosphoryl Tris was formed for every equivalent of Pi regardless of the particular ester that was hydrolyzed. The phosphoryl-enzyme theory proposes that a phosphoryl- enzyme intermediate is formed as a step in the hydrolysis of phosphate esters followed by reaction of this intermediate with water and with other phosphate acceptors to regenerate enzyme and produce Pi and transphosphorylation products of other acceptors. In this theory, the leaving group of the ester is no longer present when the reaction with Tris and with water takes place and therefore cannot influence the ratio of products. Under these circumstances, the ratio of products must be a constant, independent of the actual ester which is used as a substrate. It is also true that a different ratio of products must be obtained for each ester if these reagents react with an entity which still contains the different leaving groups. Since a constant ratio of products was obtained, it may be concluded that a phosphoryl-enzyme occurs as an intermediate in the enzymic hydrolysis of phosphate esters. © 1969, American Chemical Society. All rights reserved.